Amplite® Colorimetric Hypochlorite (Hypochlorous Acid) Assay Kit

Protocol summary

1. Prepare Hypochlorite working solution (50 µL)
2. Add Hypochlorite standards or test samples (50 µL)
3. Incubate at room temperature for 3 - 5 min
4. Monitor absorbance increase at OD of 555 ± 5 nm

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

1. Oxirite™ Hypochlorite Sensor stock solution (20X):
Add 500 µL of DMSO (Component D) into the vial of Oxirite™ Hypochlorite Sensor (Component A) to make 20X Oxirite™ Hypochlorite Sensor stock solution.

Add 250 µL of 20X Oxirite™ Hypochlorite Sensor stock solution into 5 mL of Assay Buffer (Component B), and mix well to make Hypochlorite working solution. Note: This Hypochlorite working solution is enough for one 96-well plate. It is not stable, use it promptly. Protect from light.

Table 1. Layout of Hypochlorite standards and test samples in a 96-well clear bottom microplate. H = hypochlorite standard (H1 - H7, 0.001% to 1%), BL = blank control, TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare Hypochlorite standards (H), blank controls (BL), and test samples (TS) in a 96-well clear bottom microplate as provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of Hypochlorite working solution to each well of Hypochlorite standards, blank control, and test samples to make the total Hypochlorite assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 3 - 5 minutes, protected from light.
   
   
4. Monitor the absorbance increase with an absorbance plate reader at OD = 555 ± 5 nm.